The broad long-term objective of this proposal is to provide a comprehensive description of osmoregulation in Staphylococcus aureus. It is hoped that this knowledge will have wide application to gram positive bacteria. S. aureus is an extremely halotolerant eubacterium capable of growing in media containing in excess of 3 M NaCl. The physiological mechanisms allowing this remarkable osmotolerance are poorly understood. Also, growth with NaCl is known to profoundly affect the cell physiology of S. aureus. S. aureus is a major cause of food poisoning and an extremely versatile pathogen of a variety of organs and tissues. Ability to grow in high osmolarity environments might be an important attribute of the pathogenicity of the organism. The physiology and biochemistry of osmoregulation will be studied by determining the identity and intracellular concentration of molecules (osmolytes) accumulating during growth in high osmolarity media. The dynamics of osmoregulation will be probed in experiments where cells are subjected to hyperosmotic stress. Transport systems for osmolytes will be studied in terms of their kinetics, substrate specificity and response to osmotic stress in whole cells and membrane vesicle preparations. Genetic studies will involve the creation of various osmolyte mutants by chemical and transposon mutagenesis. Osmolyte transport or metabolism mutants will be selected for inability to grow on defined medium containing 2 M NaCl plus an osmolyte (e.g., proline, glycine betaine, choline). Exogenous osmolyte-independent mutants, and proline analog-resistant transport mutants will also be created. Transposon lacZ fusion mutants will be used to study the transcriptional regulation of osmoregulatory genes. Transposon insertion mutants will be used to clone genes involved in osmoregulation.